Spore formation in the bacterium Bacillus subtilis is a relatively simple example of differentiation and involves numerous changes in cellular physiology, gene expression, and morphology. Sporulation is initiated in response to nutrient deprivation in the presence of pheromone-like factors. The goal of this research is to understand the mechanisms that control the initiation of sporulation. This involves defining the external stimuli needed to initiate spore formation, identifying the cellular components involved in signal transduction, and identifying and characterizing genes that are activated in response to sporulation conditions. Our work will focus on two regulatory genes, spoOA and spoOH, that are required for the initiation of sporulation. The spoOA gene product is homologous to other prokaryotic regulatory proteins that are involved in signal transduction. The spoOH gene product is a sigma factor of RNA polymerase that controls expression of genes needed to initiate sporulation. Specifically, we will identify and characterize genes that are controlled by these two regulatory genes. We have used a fusion-generating transposon to identify genes whose expression is dependent on spoOH, and will employ a similar strategy to identify genes that are controlled by spoOA. We will determine how these genes are expressed during growth and sporulation and the extent to which their expression depends on the spoO regulatory genes and we will characterize regulatory regions upstream of some of these genes. In addition, we will study the role of spoOA in the initiation of sporulation by continuing to isolate and characterize pseudorevertants of a spoOA missense mutation. Extragenic suppressors might identify regulatory pathways involving spoOA or proteins with which SpoOA interacts. Intragenic suppressors will be mutations that cause altered function of SpoOA, rather than loss of function. Finally, we propose to identify genes involved in the extracellular control of sporulation by isolating mutations that cause defects in the production of extracellular sporulation factors. Characterization of such mutants might facilitate the biochemical identification of these factors and will help to determine how cells sense and transduce external signals. Our studies on differentiation of this experimentally accessible microbe should provide significant insights into general mechanisms of signal transduction, gene expression, and normal and abnormal differentiation.